Proteins prepared by recombinant DNA methods are sometimes difficult to isolate because the protein must be extracted from the transformed microorganism by means such as cell lysis that typically destroy the microorganism. Obtaining sufficiently pure protein in high yield from such extraction procedures is difficult because of the large number of different organic compounds liberated when lysis takes place.
One attractive alternative to typical separation procedures is to have the host secrete the selected protein into its environment. A secreted protein is more easily separated from the culture medium, and the microorganism can survive the separation process to produce and to secrete more of the desired protein.
Most proteins naturally secreted by prokaryotes and eukaryotes are initially synthesized in the form of precursors containing an amino-terminal extension several amino acids long. This extension, called a secretion leader or signal sequence, allows the precursor protein to cross the cell membrane of the microorganism and enter the culture medium or periplasmic space of the cell. During secretion, the secretion leader is cleaved from the protein, leading to the presence of mature protein in the culture medium or periplasmic space.
Although the rate of secretion in bacteria is typically very low, bacterial secretion has been used with recombinant DNA technology. See, for example, U.S. Pat. Nos. 4,411,994 and 4,338,397, and Villa-Komaroff et al., Proc. Natl. Acad. Sci. USA, 75, 3727-31 (1978).
The general safety of yeasts and human experience with yeast fermentation have made yeasts desirable candidates for use as hosts in recombinant DNA technology. However, reported attempts to obtain secreted mature proteins from recombinant yeasts have suffered from low yields. See, for example, C. N. Chang et al., "Recognition and Cleavage of Hybrid Invertase Signals and Mature Forms of Human Interferon (IFN-.alpha.2) in Yeast," Meeting Abstracts, The Molecular Biology of Yeast, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., p. 393 (1983); B. Meyhack and A. Hinnen, "High Levels of Expression of Foreign Genes Under the Control of the Yeast PH05 Promoter," Meeting Abstracts, The Molecular Biology of Yeast, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., p. 156 (1983); S. D. Emr, "An MF.alpha.1-SUC2 (.alpha.-Factor-Invertase) Gene Fusion for Study of Protein Localization and Gene Expression in Yeast," Proc. Natl. Acad. Sci. USA, 80, pp. 7080-84 (1983); A. Brake et al., ".alpha.-Factor-Directed Synthesis and Secretion of Mature Foreign Proteins in Saccharomyces cerevisiae," Proc. Natl. Acad. Sci. USA, 81, pp. 4642-46 (1984); G. A. Bitter et al., "Secretion of Foreign Proteins from Saccharomyces cerevisiae Directed by .alpha.-Factor Gene Fusions," Proc. Natl. Acad. Sci. USA, 81, pp. 5330-34 (1984); A. Singh et al., "Synthesis, Secretion and Processing of .alpha.-Factor-Interferon Fusion Proteins in Yeast," Nucl. Ac. Res., 12, pp. 8927-38 (1984); European Patent Application 123,228; and European Patent Application 128,733; and G. Bitter et al, "Secretion of Foreign Proteins from Saccharomyces cerevisiae Directed by .alpha.-Factor Pheromone," Nucl. Ac. Res., 11, pp. 4049-63 (1983).